Compositions and methods for detecting protease activity

ABSTRACT

The invention provides a method of determining activity of a protease. The method can include the steps of (a) providing a protease substrate including a protein moiety attached to a nucleic acid moiety and a ligand moiety; (b) contacting the protease substrate with a protease under conditions wherein the protease catalyzes cleavage of the protein moiety, thereby producing a proteolytic product wherein the nucleic acid moiety is separated from at least a portion of the protein moiety and the ligand moiety; (c) contacting the proteolytic product with a receptor under conditions wherein the ligand moiety binds to the receptor to form a complex; (d) separating the complex from the nucleic acid moiety, thereby forming a separation product including the nucleic acid moiety; (e) contacting the separation product with a probe nucleic acid under conditions wherein the nucleic acid moiety hybridizes to a complementary sequence of the probe; and (f) detecting hybridization of the separation product to the probe, thereby determining activity of the protease.

This invention was made with government support under grant numberAI056869 awarded by the National Institute of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates generally to proteomics, and more specifically todetection of protease activity in formats amenable to multiplexanalysis.

Proteases are a class of enzymes with important roles in the regulationof cellular activity. Proteases act by cleaving proteins into smallerversions. This results in activation or inactivation of the proteinswhich in turn influences the role of the proteins in diversephysiological processes. Examples of physiological processes that aredirectly affected by proteases include blood coagulation, inflammation,programmed cell death, reproduction, fibrinolysis, and immune response.Numerous disease states are caused by, or can be characterized by, thealterations in the activity of specific proteases. The importance andcomplexity of the roles played by proteases in human health is evidentin the fact that there are at least 575 known and putative proteases inhumans, and it is estimated that up to 1,200 human genes encodeproteases. The ability to detect and evaluate these proteases inresearch or clinically is significant to the investigation, treatment,and management of disease states.

In addition, there are many pathogenic microorganisms that depend uponspecific protease activity for their infectivity. As an example, manyviruses depend on protease cleavage of inactive precursors, calledpro-proteins, to form active products that mediate infection. Inhibitingsuch selective cleavage can inhibit the viability of the virus. Again,the ability to evaluate these proteases in a research or clinicalsetting would benefit investigation, treatment and management of manypathogenic diseases.

Several blockbuster drugs that have been introduced to the market areprotease inhibitors. For example, protease inhibitor drugs have beendeveloped to inhibit viral proteases required for replication of HIV andin many cases have been the most effective treatments for HIV/AIDS.Other protease inhibitor drugs block the human protease, thrombin, whichis involved in blood clotting and are among the most effectivetreatments for stroke and coronary infarction. Protease inhibitor drugsare also used to treat high blood pressure. Overall, it has beenestimated that 5-10% of all pharmaceutical targets are proteases. Inthis regard, other protease inhibitors are being developed to treatparasitic, fungal, and viral infections; inflammatory, immunological,and respiratory conditions; cardiovascular and neurodegenerativedisorders including Alzheimer's disease; and cancers.

Proteases are also important in food processing Some well known examplesare the production of cheese which has traditionally relied uponproteases isolated from the stomach of unweaned calves and meattenderization which traditionally utilized papain from the leaves andunripe fruit of Carica papaya. Proteases are also used in the bakingindustry. For example, pastry dough may be prepared more quickly if itsgluten is partially hydrolyzed using a heat-labile protease that isinactivated early in the subsequent baking. Although several traditionalmethods are still practiced in food processing, there is a need for newproteases that provide more efficient food production or new varietiesand flavors. For example, the amount and types of proteases used duringcheese ripening has substantial effects on flavor and quality. As suchdifferences in proteases used for cheese ripening is largely responsiblefor the distinct varieties of cheeses available.

In a further example, proteolysis of inexpensive materials such as soyaprotein can increase the range and value of their usage in producing newfoods. In this regard, partial hydrolysis of soya protein can greatlyincrease its ‘whipping expansion’ whereas further hydrolysis can improveits emulsifying capacity. Proteases can also be used to recover proteinfrom parts of animals that would otherwise go to waste after butchering.For example, residual meat on manually butchered bones can be removed byproteases and the resulting meat slurry used to provide canned meats andsoups.

Traditionally, however proteases have not always been easy to evaluateand identify. Although specific assays have been developed to measureactivity of individual proteases, their utility for evaluating proteasesin more complex biological mixtures has been limited. Furthermore, theability to identify new substrates or inhibitors for proteases is oftendifficult using assays for individual proteases. The result isinefficient identification of useful proteases and or inhibitors.

Thus, there exists a need for assays that allow the rapid and efficientanalysis of protease activity in complex biological mixtures. There isalso a need for assays to identify previously unknown proteasesubstrates or inhibitors. The present invention satisfies these needsand provides other advantages as well.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of determining activity of a protease.The method can include the step of (a) providing a protease substrateincluding a protein moiety attached to a nucleic acid moiety; (b)contacting the protease substrate with a protease under conditionswherein the protease catalyzes cleavage of the protein moiety, therebyproducing a proteolytic product wherein the nucleic acid moiety isseparated from the protein moiety; (c) contacting the proteolyticproduct with a nucleic acid probe under conditions wherein the nucleicacid moiety hybridizes to a complementary sequence of the probe; and (d)detecting hybridization of the proteolytic product to the probe, therebydetermining activity of the protease.

The invention also provides a method of determining activity of at leastone protease. The method can include the steps of (a) providing aplurality of different protease substrates each including a proteinmoiety attached to a nucleic acid moiety, wherein the sequence of eachnucleic acid moiety is unique to the sequence of a different proteinmoiety; (b) contacting the protease substrates with at least oneprotease under conditions wherein the at least one protease catalyzescleavage of the protein moieties, thereby producing proteolytic productseach wherein the nucleic acid moiety is separated from the proteinmoiety; (c) contacting the proteolytic products each with nucleic acidprobes under conditions wherein the nucleic acid moiety hybridizes to acomplementary sequences of the probe; and (d) detecting hybridization ofthe proteolytic products to the probes, thereby determining activity ofthe at least one protease.

The invention further provides method of determining activity of aprotease. The method can include the steps of (a) providing a proteasesubstrate including a protein moiety attached to a nucleic acid moietyand a ligand moiety; (b) contacting the protease substrate with aprotease under conditions wherein the protease catalyzes cleavage of theprotein moiety, thereby producing a proteolytic product wherein thenucleic acid moiety is separated from at least a portion of the proteinmoiety and the ligand moiety; (c) contacting the proteolytic productwith a receptor under conditions wherein the ligand moiety binds to thereceptor to form a complex; (d) separating the complex from the nucleicacid moiety, thereby forming a separation product including the nucleicacid moiety; (e) contacting the separation product with a probe nucleicacid under conditions wherein the nucleic acid moiety hybridizes to acomplementary sequence of the probe; and (f) detecting hybridization ofthe separation product to the probe, thereby determining activity of theprotease.

Also provided is a method of determining activity of at least oneprotease, including the steps of (a) providing a plurality of proteasesubstrates each including a protein moiety attached to a nucleic acidmoiety and a ligand moiety, wherein the sequence of each nucleic acidmoiety is unique to the sequence of a different protein moiety; (b)contacting the protease substrates with at least one protease underconditions wherein the at least one protease catalyzes cleavage of theprotein moieties, thereby producing proteolytic products wherein thenucleic acid moieties are separated from at least a portion of theprotein moieties and the ligand moieties; (c) contacting the proteolyticproducts with at least one receptor under conditions wherein the ligandmoieties bind to the at least one receptor to form complexes; (d)separating the complexes from the nucleic acid moieties, thereby formingseparation products including the nucleic acid moieties; (e) contactingthe separation products with nucleic acid probes under conditionswherein the nucleic acid moieties hybridize to complementary sequencesof the probes; and (f) detecting hybridization of the separationproducts to the probes, thereby determining activity of the at least oneprotease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method for attaching a DNA moiety to a protein moiety

FIG. 2 shows a second method for attaching a DNA moiety to a proteinmoiety.

FIG. 3 shows a diagrammatic example of a protease substrate having anucleic acid moiety (oligonucleotide) and a protein moiety (peptide)attached via a linker having a label (tag).

FIG. 4 shows a diagrammatic example of a protease substrate having anucleic acid moiety (oligonucleotide) and a protein moiety (peptide)attached via a polyhistidine linker that serves as a label.

FIG. 5 shows a protease substrate having a protein moiety attached to aFAM label via its amino terminal residue and attached to the 5′ end of aDNA moiety via its carboxy-terminal residue; the substrate is shown in amethod in which protease activity results in decreased signal for theproteolytic product compared to the protease substrate when each isbound to a nucleic acid probe having a nucleic acid sequencecomplementary to the DNA moiety.

FIG. 6 shows a protease substrate having a protein moiety attached to abiotin label via its amino terminal residue and attached to the 5′ endof a DNA moiety via its carboxy-terminal residue, wherein the 3′ end ofthe DNA moiety is attached to a FAM label; the substrate is shown in amethod in which protease activity results in increased signal for theproteolytic product compared to the protease substrate when each isbound to a nucleic acid probe having a nucleic acid sequencecomplementary to the DNA moiety.

FIG. 7 shows a bar graph for the mean intensity of signal from acaspase-3 substrate detected on a BeadArray™ platform followingtreatment with various concentrations of enzyme approaching a limit ofdetection that is beyond 0.01 nM enzyme.

FIG. 8 shows a plot of mean intensity of signal vs. concentration forvarious caspase-3 substrates detected on a BeadArray™ platform followingtreatment in the presence or absence of caspase-3 enzyme and/orstreptavidin.

FIG. 9 shows a plot of mean intensity of signal vs. time for variousconcentrations of caspase-3 substrates detected on a BeadArray™ platformfollowing treatment with caspase-3 enzyme then streptavidin-basedextraction.

FIG. 10 shows a plot of mean intensity of signal vs. time for acaspase-3 substrate detected on a BeadArray™ platform followingtreatment with caspase-3 enzyme and various concentrations of caspase-3inhibitor then streptavidin-based extraction.

FIG. 11 shows a plot of mean intensity of signal vs. concentration forvarious caspase-3 substrates detected at specific probes on a BeadArray™platform for individual reactions of each substrate with caspase-3enzyme followed by streptavidin-based extraction.

FIG. 12 shows a plot of mean intensity of signal vs. concentration forvarious caspase-3 substrates detected at specific probes on a BeadArray™platform for a multiplex reaction in which all three substrates wereincubated simultaneously and in the same tube with caspase-3 enzymefollowed by streptavidin-based extraction.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides compositions and methods for evaluating proteaseactivity. The methods are well suited for multiplex analysis ofpluralities of proteases, protease substrates or protease inhibitors orcombinations thereof. However, if desired the methods or compositionscan be used in a singleplex format (i.e. using a single species ofprotease and a single species of protease substrate). An advantage ofthe invention is that the use of a multiplex format allows for increasedefficiency and reduced reagent consumption for determining proteaseactivity. Moreover, the use of a multiplex format can allow convenientevaluation of the effects of complex mixtures on protease activity andcan, therefore, more appropriately simulate the complex mixtureexperienced by proteases in their natural milieu compared to the use ofmost currently available protease assays.

Definitions

As used herein, the term “protease” is intended to mean an agent thatcatalyzes the cleavage of peptide bonds in a protein. A protease canhave sequence specificity, splitting a peptide bond of a protein basedon the presence of a particular amino acid sequence in the protein.However, non-sequence specific proteases are also useful. A protease canbe characterized according to the location in a protein where itcleaves, an endoprotease cleaving a protein between internal amino acidsof an amino acid chain and an exoprotease cleaving a protein to removean amino acid from the end of an amino acid chain. A protease can becharacterized according to mechanism of action, being identified, forexample, as a serine protease, cysteine (thiol) protease, aspartic(acid) protease, metalloprotease or mixed protease depending on theprincipal amino acid participating in catalysis. A protease can also beclassified based on the action pattern, examples of which include anaminopeptidase which cleaves an amino acid from the amino end of aprotein, carboxypeptidase which cleaves an amino acid from the carboxylend of a protein, dipeptidyl peptidase which cleaves two amino acidsfrom an end of a protein, dipeptidase which splits a dipeptide andtripeptidase which cleaves an amino acid from a tripeptide. Typically, aprotease is a protein enzyme. However, non-protein agents capable ofcatalyzing the cleavage of peptide bonds in a protein, especially in asequence specific manner are also useful in the invention.

As used herein, the term “activity,” when used in reference to aprotease, is intended to mean binding of the protease to a proteasesubstrate or hydrolysis of the protease substrate or both. The activitycan be indicated, for example, as binding specificity, catalyticactivity or a combination thereof. The activity of a protease can beidentified qualitatively or quantitatively in accordance with thecompositions and methods disclosed herein. Exemplary qualitativemeasures of protease activity include, without limitation,identification of a substrate cleaved in the presence of the protease,identification of a change in substrate cleavage due to presence ofanother agent such as an inhibitor or activator, identification of anamino acid sequence that is recognized by the protease, identificationof the composition of a substrate recognized by the protease oridentification of the composition of a proteolytic product produced bythe protease. Activity can be quantitatively expressed as units permilligram of enzyme (specific activity) or as molecules of substratetransformed per minute per molecule of enzyme (molecular activity). Theconventional unit of enzyme activity is the International Unit (IU),equal to one micromole of substrate transformed per minute. A proposedcoherent Système Internationale (SI) unit is the katal (kat), equal toone mole of substrate transformed per second.

As used herein the term, “protease substrate” is intended to mean amolecule that can be cleaved by a protease. A protease substrate istypically a protein or protein moiety having an amino acid sequence thatis recognized by a protease. A protease can recognize the amino acidsequence of a protease substrate due to the specific sequence of sidechains or due to properties generic to proteins. A protease substratecan also be a protein mimetic or non-protein molecule that is capable ofbeing cleaved or otherwise covalently modified by a protease.

As used herein, the term “proteolytic product” is intended to mean atleast one molecule that has been cleaved by a protease. A protease canproduce one, two or more proteolytic products. For example, twomolecules can be produced when a molecule is cleaved by a protease andthe portions on either side of the protease cleavage site are nototherwise tethered to each other. A single molecule can result in caseswhere portions on either side of a cleaved site are tethered to eachother, for example, via a disulfide linkage or non-covalent interaction.Several products can result when a protein or protein moiety is cleavedat several locations and the portions on either side of the proteasecleavage sites are not otherwise tethered to each other.

As used herein, the term “level,” when used in reference to a reactionproduct, is intended to mean an amount of the reaction product or of acharacteristic of the reaction product. An amount of a reaction productor characteristic of the reaction product can be expressed in absoluteterms or relative terms. Exemplary absolute amounts include, but are notlimited to, number of molecules, mass, concentration, catalyticactivity, generated signal such as an optical signal, or other amountsknown in the art and measurable by known methods. Exemplary relativeamounts include, without limitation, a ratio of the foregoing amountsfor two different reaction products or two different populations ofreaction products. The characteristic of a reaction product can bedetected by methods described herein or otherwise known in the art suchas optical spectroscopy, mass spectroscopy, detection of magnetic orelectronic properties or calorimetry.

As used herein, the term “protein” is intended to mean a chain of aminoacids connected by peptide bonds. The term is intended to include chainshaving any possible number of amino acids, unless explicitly indicatedotherwise. Accordingly a protein can include a single linear chainhaving at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100,1000, 10,000, 100,000 or more amino acids. If desired, a protein usefulin the invention can have a maximum length including, for example, atmost about 100,000, 10,000, 1000, 100, 10, 5 or fewer in a linear chain.A protein can include one or more of the 20 amino acids used by a humancell to translate RNA into protein. Furthermore, a protein can includeother amino acids such as non-naturally occurring amino acids.

A protein when included as part of a larger molecule or substrate isreferred to herein as a “protein moiety.” For example, a protein moietycan be covalently attached to another protein moiety or to a moietyhaving other compositions such as a nucleic acid, polysaccharide,phosphate, isoprenyl group or enzyme cofactor. A protein moiety can beattached to another protein moiety via a peptide bond to form a linearchain. Alternatively or additionally, a protein moiety can be attachedto another protein moiety to form a branched chain, for example, via thethioether linkage of cystine or via an isopeptide bond such as thatformed between the alpha carboxy of ubiquitin and the epsilon aminogroup in the side chain of a lysine residue. A protein moiety can benon-covalently attached with another moiety or molecule such as a secondprotein or a nucleic acid. A species of proteins or protein moieties isunderstood to be a group that all include the same sequence of aminoacids

As used herein, the term “nucleic acid” is intended to mean polymermolecule composed of subunits having purine or pyrimidine bases. Anucleic acid when included as part of a larger molecule is referred toherein as a “nucleic acid moiety.” A nucleic acid useful in the presentinvention will generally contain phosphodiester bonds, and can include,for example, DNA or RNA. If desired to suit a particular application,DNA or RNA analogs having alternate backbones can be used, including,for example, phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925(1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970);Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl.Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984),Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al.,Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., NucleicAcids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989),O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), or peptidenucleic acid linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992);Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature,365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which areincorporated by reference). Other polynucleotide analogs include thosewith positive backbones (Denpcy et al., Proc. Nat Acad. Sci. USA 92:6097(1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem.Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc.110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars can also be used in the invention (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several other nucleic acidanalogs are described in Rawls, C & E News Jun. 2, 1997 page 35.

A nucleic acid or nucleic acid moiety can be single stranded, doublestranded or contain portions of both double stranded and single strandedsequence. A polynucleotide can be DNA, such as genomic DNA (gDNA) orcopy DNA (cDNA); RNA such as messenger RNA (mRNA), transfer RNA (tRNA)or ribosomal RNA (mRNA); or a hybrid containing any combination ofdeoxyribo- and ribo-nucleotides or any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthanine,hypoxanthanine, isocytosine, isoguanine, or the like. A “species” ofnucleic acids or nucleic acid moieties is understood to be a group thatall include the same nucleic acid sequence.

As used herein, the term “label moiety” is intended to mean one or moreatoms that can be specifically detected to indicate the presence of asubstance to which the one or more atom is attached. A label moiety canbe a primary label that is directly detectable or secondary label thatcan be indirectly detected, for example, via interaction with a primarylabel. Exemplary primary labels include, without limitation, an isotopiclabel such as a naturally non-abundant heavy isotope or radioactiveisotope, examples of which include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ³²P, ³⁵Sor ³H; optically detectable moieties such as a chromophore, luminophore,fluorophore, quantum dot or nanoparticle; electromagnetic spin label;calorimetric agent; magnetic substance; electron-rich material such as ametal; electrochemiluminescent label such as Ru(bpy)₃ ²⁺; moiety thatcan be detected based on a nuclear magnetic, paramagnetic, electrical,charge to mass, or thermal characteristic; or light scattering orplasmon resonant materials such as gold or silver particles.Fluorophores that are useful in the invention include, for example,fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, fluorescein isothiocyanate, carboxyfluorescein(FAM), dichlorotriazinylamine fluorescein, rhodamine,tetramethylrhodamine, umbelliferone, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, LuciferYellow, Cascade Blue™ Texas Red, alexa dyes, dansyl chloride,phycoerythin, green fluorescent protein and its wavelength shiftedvariants, bodipy, and others known in the art such as those described inHaugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; TheSynthegen catalog (Houston, Tex.), Lakowicz, Principles of FluorescenceSpectroscopy, 2nd Ed., Plenum Press New York (1999), or WO 98/59066.

Exemplary secondary labels that can be used in the invention include,without limitation, a binding moiety such as a receptor, ligand or othermember of a pair of molecules having binding specificity for each other.Exemplary binding moieties having specificity for each other include,without limitation, streptavidin & biotin, avidin & biotin or an antigen& antibody such as rabbit IgG & anti-rabbit IgG. Specific affinitybetween two binding partners is understood to mean preferential bindingof one partner to another compared to binding of the partner to othercomponents or contaminants in the system. Binding partners that arespecifically bound typically remain bound under the detection orseparation conditions described herein, including wash steps to removenon-specific binding. Depending upon the particular binding conditionsused, the dissociation constants of the pair can be, for example, lessthan about 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹²M⁻¹. Secondary labels also include enzymes or their substrates, whereinthe combination produces a detectable product, examples of which includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase which produce colorimetric products using colorreagents that are commercially available, for example, fromSigma-Aldrich (St. Louis, Mo.) or Invitrogen (Carlsbad, Calif.).

The terms “receptor” and “ligand” are used herein for semantic clarityin identifying binding partners and are intended to be interchangeable,unless explicitly indicated to the contrary. Accordingly, the term“receptor” is intended to mean a molecule that is capable of selectivelybinding a ligand and the term “ligand” is intended to mean a moleculethat is capable of selectively binding a receptor. The terms areintended to encompass receptors or ligands that have other functions aswell. However, the terms are not intended to be limited by any otherfunction unless indicated otherwise. For example, a receptor can be anaturally occurring polypeptide having signal transducing activity or afunctional fragment thereof that exhibits selective binding to a ligandwhether or not the functional fragment has signal transducing activity.

As used herein, the term “array” refers to a population of differentprobe molecules that are attached to one or more substrates such thatthe different probe molecules can be differentiated from each otheraccording to their relative location. An array can include differentprobe molecules that are each located at a different addressablelocation on a substrate. Alternatively, an array can include separatesubstrates each bearing a different probe molecule, wherein thedifferent probe molecules can be identified according to the locationsof the substrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid such as a fluidstream.

Description of Particular Embodiments

The invention provides a method of determining activity of a protease.The method can include the steps of (a) providing a protease substratehaving a protein moiety attached to a nucleic acid moiety; (b)contacting the protease substrate with a protease under conditionswherein the protease catalyzes cleavage of the protein moiety, therebyproducing a proteolytic product wherein the nucleic acid moiety isseparated from the protein moiety; (c) contacting the proteolyticproduct with a nucleic acid probe under conditions wherein the nucleicacid moiety hybridizes to a complementary sequence of the probe; and (d)detecting hybridization of the proteolytic product to the probe, therebydetermining activity of the protease.

A method of the invention can be used to determine a protease activitythat is indicative of the presence of the protease in a sample.Accordingly, the invention can be used to determine the presence orabsence of a protease in a sample. For example, a protease substratehaving a known or determinable amino acid sequence can be contacted witha sample in a method of the invention and cleavage of the substratedetected. Cleavage, thus, indicates that the sample includes a proteasethat recognizes the substrate.

An activity of a protease determined in a method of the invention caninclude one or more characteristic of the protease. For example, theamino acid sequence of a substrate cleaved in a method of the inventioncan be determined and, if desired, the location where cleavage occurscan also be identified. Such evaluations can be used to determinespecificity of a protease for a particular amino acid sequence. Theamino acid recognition sequence identified in a method of the inventioncan be a discreet sequence or a consensus sequence, having one or moredegenerate position. Other characteristics of a protease that can bedetermined in a method of the invention include, for example, rate ofcatalysis, binding affinity for a particular amino acid sequence oreffects of particular conditions or agents on catalytic or bindingactivity of the protease. Exemplary conditions or agents that can beevaluated include activators, inhibitors, other proteases, pH, salt,temperature or the like.

Activity of a protease can be determined qualitatively or quantitativelyin a method of the invention. Exemplary qualitative determinations caninclude, without limitation, identification of substrate specificity;identification of conditions that increase or decrease activity;determination of the presence or absence of activators or inhibitors ofthe protease; or determination of relative activities between differentsamples, proteases, substrates or conditions Quantitative determinationscan be made for these or other characteristics for a more precisemeasure, if desired. For example, a method of the invention can be usedto determine the binding affinity of a protease for a particularsubstrate in the form of a thermodynamic constant, such as adissociation constant. Accordingly, the methods can be used to identifya protease having dissociation constant for a particular substrate thatis, for example, less than about 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M⁻¹. Similarly, an inhibition constant can bedetermined for a particular inhibitor in the presence of a protease andits substrate. A further quantitative measure that can be determined ina method of the invention is a catalytic rate constant for proteincleavage. Such kinetic and thermodynamic constants can be determinedusing titration measurements and/or time dependent measurements inaccordance with analyses known in the art as described, for example, inSegel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium andSteady-State Enzyme Systems, Wiley, John & Sons, Incorporated (1994).

A method of the invention can be used to determine activity for asequence-specific protease that recognizes a particular amino acidsequence and cleaves a protein having that sequence as set forth above.Alternatively or additionally, the invention can be used to determineactivity for a non-sequence specific protease that is promiscuous withregard to the amino acid sequences it will recognize and cleave.Proteases, whether specific or promiscuous, are involved in severalimportant physiological processes. By providing for determination ofprotease activity the invention further allows for identification orcharacterization of one or more physiological processes involving theprotease. For example, determination of a protease activity involvedwith pathogenesis can provide valuable information for identifying orcharacterizing host-pathogen interactions. Such information can lead todiagnostic or prognostic information. Further examples of physiologicalprocesses that can be characterized or identified using a method of theinvention include, but are not limited to, blood coagulation,inflammation, programmed cell death (apoptosis), reproduction,fibrinolysis, cancer, the cell cycle, transcriptional regulation, thesecretory pathway, cellular stress response or immune response (forexample, antigen presentation by MHC molecules during the T-cell immuneresponse).

The ability to perform a multiplexed protease assay in accordance withthe invention provides an advantage for the evaluation of proteasecascades, signal transduction cascades or other biochemical pathwaysthat are influenced by multiple proteases. A multiplexed protease assaycan more closely mimic the complexities of biological systems such thatevaluation of the results can yield observations and information that isdifferent or absent when the results of individual assays, using thecomponents of the multiplexed assay, are evaluated alone or incombination.

A substrate useful in the invention can have a protein moiety attachedto a nucleic acid moiety in any of a variety of configurations wherein aprotease is capable of cleaving the protein moiety. In particularembodiments, a protein moiety is attached to a nucleic acid moiety in alinear arrangement such that the nucleotide at the 3′ or 5′ end of thenucleic acid moiety is attached to the amino acid at the carboxy oramino terminal residue of the protein moiety. Alternatively, the twomoieties can be attached such that an internal amino acid of the proteinmoiety is attached to the nucleic acid moiety and/or an internalnucleotide of the sequence of the nucleic acid moiety is attached to theprotein moiety.

Attachment of a protein moiety and nucleic acid moiety is typicallymediated by at least one covalent bond. An attaching bond can be made toany desired portion of a protein moiety including, without limitation, abackbone carbon, nitrogen or oxygen or a sidechain (“R”) group.Similarly, a bond can be made to any desired portion of the nucleic acidmoiety including, but not limited to, the backbone or base. In theexemplary case where DNA or RNA is used, an attaching bond can be madeto the phosphodiester backbone, the sugar moiety or the base. If DNA orRNA analogs, such as those set forth above, are used then a covalentbond used for attachment can be made to known structural moietiestherein. Attachment of a PNA-based nucleic acid moiety to a proteinmoiety is particularly convenient since both moieties contain a similarbackbone structure. More specifically, the two moieties can be attachedvia a peptide bond between the terminal alpha carbonyl of one moiety andterminal alpha amino of the other moiety. PNA or peptide-PNA chimaerascan be synthesized using methods known in the art as described, forexample, in U.S. Pat. No. 6,713,602 or Nielsen et al. Science254:1497-1500 (1991).

Exemplary methods for attaching a DNA moiety to a protein moiety areshown in FIGS. 1 and 2. A DNA molecule having a benzaldehyde nucleotideat the 3′ end can be synthesized using methods known in the art such asthose described in U.S. Ser. No. 10/739,959. The benzaldehyde residue onthe DNA can be coupled to a hydrazine moiety on the amino terminus of aprotein to form a hydrazone bond as shown in FIG. 1. Coupling to formthe hydrazone bond can be carried out at pH between 4.0 and 7.0. In analternative method, the benzaldehyde residue on the DNA can be coupledto an aminooxyacetic moiety on the amino terminus of a protein to forman oxime bond as shown in FIG. 2. Coupling to form the oxime bond can becarried out at pH between 4.0 and 5.5. The locations for the reactivemoieties on the nucleic acid and protein reactants are provided forpurposes of illustration. It will be understood that in order toaccommodate various uses of the reagents, the reactive moieties can beplaced at locations other than those shown in the Figures or that thereagents can be swapped with each other such that, for example, theprotein contains a benzaldehyde moiety and the nucleic acid contains anaminooxyacetic moiety. Furthermore other reactive aldehydes,hydroxylamines and/or hydrazines can be used in place of thoseexemplified above.

Other pairs of reactive moieties that can be used to couple a proteinand nucleic acid molecule to form a protein-nucleic acid conjugateinclude, for example, Thiol and bromoacetyl, which can be reacted toform a thialkyl linkage; thiol and maleimide which can be reacted toform a maleimide-thioalkyl linkage; aldehydes and cysteine, which can becoupled to form a triazolidine linkage, for example, at pH 2.0 to 8.0;aldehydes and serine, which can be coupled to form an oxazolidinelinkage. These and other methods that can be used to synthesize nucleicacid-protein conjugates are described, for example, in Zubin et al.,Russian Chemical Reviews 71:239-264 (2002) or Tung et al., BioconjugateChemistry 11:605-618 (2000).

In the exemplary substrate configurations shown in FIGS. 1 and 2,separate label moieties are attached to the ends of the protein moietyand nucleic acid moiety, respectively. As set forth in further detailbelow, a label can be attached to a portion of a nucleic acid-proteinconjugate in a configuration that allows the label to be separated fromthe nucleic acid moiety upon proteolysis of the conjugate. Alternativelyor additionally, a label can be attached to a portion of a nucleicacid-protein conjugate in a configuration that allows the label to beseparated from a portion of the protein moiety upon proteolysis of theconjugate. For example, an alternative configuration can include aprotein moiety having two attached labels, wherein a first label isattached to a first location of the protein moiety and a second labelattached to a second location of the protein moiety and a site in theprotein moiety that is capable of being cleaved by a protease occursbetween the locations of the two labels. In this case, a nucleic acidmoiety can be attached to the protein moiety such that the location ofthe first label occurs between the nucleic acid moiety and the proteaserecognition site. Cleavage of the nucleic acid-protein conjugate at theprotease recognition site will result in a first fragment containing thenucleic acid moiety and first label, and a second fragment containing aportion of the protein moiety and the second label.

Attachment between a nucleic acid moiety and protein moiety can also bemediated by non-covalent bonds. For example, each moiety can include apartner capable of forming a receptor-ligand complex such as avidin &biotin or other pairs known in the art such as those set forthpreviously herein.

Attachment between the protein and nucleic acid moieties of a substratecan be mediated by a linker that further includes a primary or secondarylabel. The use of a linker having a label can provide the non-limitingadvantage of allowing detection of the substrate or isolation of thesubstrate, for example, during synthesis. In a particular embodiment, alinker can include a label that is produced when an appropriatesubstrate is synthesized. If reactants, undesired reaction side productsor both lack the label then the label can be used in order to separatethe desired product from other reaction components or to monitorreaction progress or yield. A diagrammatic example of a substrate havinga nucleic acid moiety (oligonucleotide) and a protein moiety (peptide)attached via a linker having a label (tag) is shown in FIG. 3. As shownin the figure, unreacted oligonucleotide and peptide reagents haveportions of the tag that are not functional. However, correctconjugation to form the desired substrate yields a product having afunctional tag that can be used to detect the substrate or isolate itfrom other reaction components.

A first example of a substrate having a labeled linker and its use forseparating the substrate from a reaction mixture is shown in FIG. 4. Asshown in the figure, the nucleic acid (oligonucleotide) and protein(peptide) moieties each contain a trihistidine sequence. Upon successfulconjugation, the trihistidines are directly linked to form a substratehaving a nucleic acid moiety and a protein moiety attached via ahexahistidine linker. Isolation of the substrate from other reactioncomponents can be achieved using solid-phase extraction orchromatography with immobilized metal ion media such as Ni²⁺ Sepharose.In this case, separation can occur by exploiting the substantiallystronger affinity of Ni²⁺ Sepharose for hexahistidine compared totrihistidine. Similarly, reactants having dihistidines can be used suchthat the reaction product forms a tetrahistidine label that can beseparated from reactants based on higher affinity of Ni²⁺ Sepharose fortetrahistidine compared to dihistidine. Separation of substrates basedon the presence of polyhistidine labels can be carried out using methodsknown in the art as described, for example, in U.S. Pat. No. 4,569,794,5,310,663, 4,877,830, 5,047,513, or 5,284,933; Hochuli et al., J.Chromatography 411:177-184 (1987); or product literature from suppliersof histidine tag reagents such as Qiagen.

Other protein based labels that can be used in the invention, such as ina linker include, for example, Arg-tag, calmodulin-binding peptide,cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase,FLAG-tag, HAT-tag, maltose-binding protein, NusA, S-tag, SBP-tag,Strep-tag, and thioredoxin. Protein and nucleic acid reactants to beconjugated can each have inactive portions of the above tags such that acomplete functional label is present in the properly conjugated linker.The resulting labels can be used under separation or detectionconditions known in the art as described, for example, in Terpe, Appl.Microbiol. Biotechnol. 60:523-533 (2003). Furthermore, those skilled inthe art will recognize that such labels can be used as secondary labelsin other embodiments set forth herein such as detection of substrates inan array or other format.

Other labels that can be used in the invention, for example, in a linkerbetween a protein moiety and nucleic acid moiety include, for example, anucleic acid sequence or an epitope for an antibody. In embodimentswherein a nucleic acid-based label is used, detection can be carried outusing a complementary probe having higher affinity for the completelabel sequence when present in the joined linker compared to itscomponent portions present separately in the protein and nucleic acidmoieties. Exemplary epitopes that can be useful include, withoutlimitation, a nucleic acid sequence containing inosine or other baseanalog or an amino acid sequence having a non-natural amino acid ormodification. Again, precursors of the label can be present on reactantssuch that a desired product having a protein moiety and nucleic acidmoiety attached by a linker containing the label can be detected orisolated via the label.

A protease and protease substrate can be contacted under conditionswherein the protease cleaves the substrate. Typically, the reaction iscarried out in an aqueous solvent. Protease and protease substrate canbe added to a reaction vessel in any order, for example, the proteasecan be added to a reaction vessel containing the substrate, thesubstrate can be added to a reaction vessel containing the protease orboth can be added simultaneously. Accordingly, addition of one or morecomponents to a protease reaction can be used to initiate the reactionat a defined time. This can be particularly useful for time-basedmeasurements such as those used in kinetic assays. It will be understoodthat addition of a component to a reaction can include initial physicalcontact of the component with other components of the reaction oractivation of the component from an inactive or sequestered state. Forexample, a reaction component, such as a substrate or protease, can bepresent in a caged state by sequestration with another agent. As afurther example, a reaction component can be in a non-reactive form andconverted to an active form to initiate a reaction. For example, apro-protease, having a terminal inhibitory sequence, can be initiallypresent in a reaction vessel and then activated by removal of theinhibitory sequence to initiate the protease reaction. Furthermore, areaction component, such as a protease or substrate, can be activated byaddition or removal of a modification such as a phosphate, methyl,acetyl, oligosaccharide, sulfate, poly(ADP-ribose), or isoprenyl (forexample, farnesyl or geranylgeranyl) moiety. Accordingly, a method ofthe invention can be used to determine the effect of such modificationson protease activity, or in turn, to identify agents that altermodification state of a protease or protease substrate.

Components other than protease and substrate can be present in areaction if desired including, for example, a pH buffer, salt, reducingagent, agent for increasing viscosity or protease inhibitor. A proteaseinhibitor can be present for the purpose of determining its effect onthe activity of a protease of interest with a particular substrate.Alternatively, an inhibitor can be present in a reaction to preventunwanted activity of one or more proteases other than the protease ofinterest. Furthermore, conditions can be selected to favor specificityof a protease of interest for a particular substrate or to increaseoverall activity of the protease for several substrates. Those skilledin the art will know or be able to determine appropriate conditions forprotease cleavage according to that which is known in the art asdescribed, for example, in Coligan et al., Current protocols in ProteinScience, John Wiley and Sons, Baltimore, Md. (2000) or Barrett et al.,Handbook of Proteolytic Enzymes, Elsevier, Amsterdam, The Netherlands(2004).

A method of the invention can include one or more steps that are carriedout using liquid-phase or solid-phase conditions or both. Morespecifically, a protease and substrate can be contacted under aliquid-phase or solid-phase conditions or both. As an example of aliquid-phase step, a soluble protease can be contacted with a solubleprotease substrate under conditions wherein the substrate is cleaved bythe protease. A solid-phase step can then ensue, such as capture of thesubstrate or a proteolytic product of the reaction on a solid-phasesupport. The solid-phase support can have a receptor that is specificfor the substrate, proteolytic product or both. If desired, substrateand proteolytic product can be captured by different receptors that arespatially distinguishable on one or more solid supports. In a furtherexemplary embodiment, a soluble protease can be contacted with aprotease substrate attached to solid-phase support under conditionswherein the substrate is cleaved and soluble proteolytic product isreleased. The proteolytic product can include a label such thatproteolysis is detected as a loss of signal as set forth in furtherdetail below. In particular embodiments, the proteolytic product can bedetected in solution or by capture with a second solid-phase support. Itis also possible to attach, on a solid-phase support, a protease orother reaction component described herein and use the support in amethod of the invention.

In embodiments, including attachment of a protease substrate or otheragent to a solid support, the solid support can be selected, forexample, from those described herein with respect to detection arrays.Particularly useful substrates include, for example, magnetic beadswhich can be easily introduced to a reaction mixture and easily removedwith a magnet. Other known affinity chromatography substrates can beused as well. Known methods can be used to attach a nucleic acid orprotein moiety to a solid support including, for example, this describedin U.S. patent application Ser. Nos. 10/651,568 or 10/739,959, WO01/41918, or WO 04/001646.

A nucleic acid probe can be contacted with a nucleic acid moiety for asubstrate or its proteolytic product under conditions wherein thenucleic acid moiety hybridizes to the probe. A variety of hybridizationconditions can be used in the present invention, such as high, moderateor low stringency conditions including, but not limited to thosedescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Brentet al., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (2003). Stringent conditions favor specificsequence-dependent hybridization. In general, longer sequences andincreased temperatures favor specific sequence-dependent hybridization.A useful guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993).

A probe used in a method of the invention can include a complementarysequence that is any length capable of binding to a nucleic acid moiety.The complementary sequences can include all or a portion of the probe ornucleic acid moiety. Relatively short complementary sequences can beuseful including, for example, those that are at most 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80,90, or 100 nucleotides in length. Those skilled in the art willrecognize that specificity of hybridization is generally increased asthe length of the complementary sequences is increased. Accordingly,complementary sequences used in a method of the invention can be atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, or 100 or more nucleotides long. Thoseskilled in the art will recognize that a nucleic acid primer used in theinvention can also have any of the exemplary lengths set forth above.However, probes and primers useful in the invention can have lengthsother than those exemplified above, if desired.

Hybridization steps used in the invention are generally carried outunder stringency conditions which selectively allow formation of ahybridization complex in the presence of complementary sequences.Stringency can be controlled by altering a step parameter that is athermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration, pH, organic solvent concentration, or the like. Theseparameters can also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, if desired, certainsteps can be performed under relatively high stringency conditions toreduce non-specific binding.

Generally, high stringency conditions include temperatures that areabout 5-10° C. lower than the thermal melting point (T_(m)) for theannealing sequences at a particular ionic strength and pH. Highstringency conditions include those that permit a first nucleic acid tobind a complementary nucleic acid that has at least about 90%complementary base pairs along its length and can include, for example,sequences that are at least about 95%, 98%, 99% or 100% complementary.Stringent conditions can further include, for example, those in whichthe salt concentration is less than about 1.0 M sodium ion (or othersalts), typically about 0.01 to 1.0 M concentration at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short annealing sequences(e.g. 10 to 50 nucleotides) and at least about 60° C. for long annealingsequences (e.g. greater than 50 nucleotides). High stringency conditionscan also be achieved with the addition of helix destabilizing agentssuch as formamide. High stringency conditions can include, for example,conditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Nucleic acid hybrids can be further stabilized bycovalent linkage between strands using one or more cross-linking agents.

Moderately stringent conditions include those that permit a firstnucleic acid to bind a complementary nucleic acid that has at leastabout 60% complementary base pairs along its length. Depending upon theparticular conditions of moderate stringency used, a hybrid can formbetween sequences that have complementarity for at least about 75%, 85%or 90% of the base pairs along the length of the hybridized region.Moderately stringent conditions include, for example, conditionsequivalent to hybridization in 50% formamide, 5× Denhart's solution,5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS,at 65° C.7

Low stringency hybridization includes, for example, conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE are well known to those of skill inthe art as are other suitable hybridization buffers (see, for example,Sambrook et al., supra (2001) or Brent et al., supra (2003)).

A probe can be hybridized to a nucleic acid moiety of a proteasesubstrate or proteolytic product to distinguish the substrate orproteolytic product from other components of a proteolysis reaction. Theprobe can be distinguished based on known or determinablecharacteristics such as spatial or temporal location in a collection ofprobes or other environment, nucleic acid sequence, presence of aparticular label or the like. For example, a probe can be attached to asolid-phase support such that the location of the solid-phase support orlocation of the probe on the solid-phase support can be used todistinguish the protease substrate or proteolytic product. For example,a solid-phase particle having an attached probe can be distinguishedaccording to its location on a surface such as an array of particles oraccording to its location in a fluid stream such as in a flow cytometer.In another example, a probe attached at a discrete location on asolid-phase support, such as an array of probes, can be distinguishedaccording to its location on the solid-phase support. In the foregoingexamples, a protease substrate or proteolytic product of the inventioncan be distinguished according to its interaction with a particularprobe. Such interactions can be detected according to methods set forthin detail below, such as detection of a label added to the probe,protease substrate or proteolytic product of a hybrid or detection of aphysical property of such a hybrid.

A label moiety can be attached to a protease substrate of the inventionin a configuration that allows the substrate to be distinguished from aproteolytic product of the substrate. For example, when a substratehaving a nucleic acid moiety and protein moiety is used in a method ofthe invention, a label moiety can be attached to the protein moiety suchthat proteolysis of the substrate produces a proteolytic product whereinthe nucleic acid moiety is separated from the label moiety. Proteaseactivity can thus be determined based on detection of the nucleic acidmoiety absent the label. An exemplary embodiment is shown in FIG. 5 inwhich the amino terminal end of a protein moiety is attached to a FAMlabel and the carboxy-terminal end of the protein moiety is attached tothe 5′ end of a DNA moiety. As shown in the Figure, protease activityresults in separation of the FAM label from the fragment having the DNAmoiety. Thus, protease activity can be determined according to anabsence of signal or loss of signal on a solid-phase attached probe thatis complementary to the nucleic acid moiety. It will be understood thatother nucleic acids, labels or both, such as those set forth elsewhereherein, can be attached to a protein moiety in the orientation shown inFIG. 5.

Although the example of FIG. 5 describes a protein moiety having anucleic acid moiety and label moiety attached at either end, it will beunderstood that the nucleic acid moiety, label or both can be attachedat different locations along the length of the protein moiety. In suchembodiments, the substrate and product can be distinguished so long asprotease activity separates the nucleic acid and label moieties.Furthermore, another nucleic acid moiety, label or both can be used in aprotease substrate having the configuration exemplified in FIG. 5.

In particular embodiments, a label moiety can be attached to the nucleicacid moiety of a protease substrate. The label can be used to determinewhether or not the nucleic acid moiety has attached to a complementarynucleic acid probe, for example, as a control to evaluate hybridizationconditions. Such a substrate having a label moiety attached to thenucleic acid moiety can also be useful to determine whether or notproteolysis has occurred by determining whether or not the label ispresent on a receptor, such as an antibody, that binds to the proteinmoiety. For example, both the antibody and nucleic acid moiety can beattached to different label moieties such that the presence of bothlabels indicates absence of proteolytic activity whereas the presence ofthe antibody-attached label moiety and absence of the nucleicacid-attached label moiety indicates presence of proteolytic activity.Similarly, presence or absence of the nucleic acid-attached label moietyon a particular antibody bearing particle or at a particular location onan antibody bearing surface can be detected to determine absence orpresence of proteolytic activity, respectively. Although the examplesabove and elsewhere in the specification refer to absence of proteolyticactivity, it will be understood that absence of proteolysis can becharacterized by low levels of substrate cleavage so long as they arenot substantial or statistically relevant compared to levels detected orexpected for actual proteolytic activity. Furthermore, methods similarto those exemplified above can be used to determine a decrease inproteolytic activity, for example, in the presence of a proteaseinhibitor.

As set forth above and elsewhere herein, a label moiety that is not anatural component of nucleic acids or proteins found in nature, thusexogenous to these molecules, can be used in a method of the invention.However, it will be understood that a substrate and/or proteolyticproduct used in a method of the invention need not have an exogenouslabel moiety. In an embodiment wherein a substrate having a nucleic acidmoiety and protein moiety is used in a method of the invention, theprotease substrate can be distinguished from the proteolytic product dueto the presence or absence, respectively, of a receptor that binds theprotein moiety when the nucleic acid moiety is hybridized to acomplementary nucleic acid probe. A particularly useful receptor is anantibody that binds specifically to the amino acid sequence of theproteolytic substrate. Alternatively, presence or absence of the proteinmoiety can be determined based on chemical detection methods including,for example, non-specific chemical methods such as ninhydrin staining orcolorometric stains like Coomassie blue or silver stain. If desired,sequence-specific chemical methods such as amino acid sequencing can beused to detect presence or absence of a protein moiety in a proteasesubstrate or proteolytic product.

Exemplary primary and secondary labels that can be used in a method ofthe invention are set forth above in the definitions section. Otherlabels can be used as well. Secondary labels can be useful for attachinga protease substrate or proteolytic product or other agent to asolid-phase support. Secondary labels that can be used include receptorsor ligands. Exemplary pairs of ligands and receptors that can be used inthe invention include, without limitation, antigen & immunoglobulin oractive fragments thereof, such as FAbs; immunoglobulins from differentorganisms or of different subtype that bind each other (or activefragments); avidin & biotin, or analogs thereof having specificity foravidin such as imino-biotin; streptavidin & biotin, or analogs thereofhaving specificity for streptavidin; complementary nucleic acidmolecules; or carbohydrates & lectins. It will be understood that eitherpartner in the above-described pairs can be attached to a proteasesubstrate, proteolytic product solid-phase support or other agent usefulin the invention. It will be further understood that several usefullabel moieties can function as both primary and secondary labels in amethod of the invention. For example, strepatvidin-phycoerythrin can bedetected as a primary label due to fluorescence from the phycoerythrinmoiety or it can be detected as a secondary label due to its affinityfor anti-streptavidin antibodies.

In a particular embodiment, a secondary label can be a chemicallymodifiable moiety. In this embodiment, labels having reactive functionalgroups can be incorporated into a protease substrate, proteolyticproduct or other agent useful in the invention. The functional group canbe subsequently covalently reacted with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups.

Secondary labels can be particularly useful when attached to proteasesubstrates or proteolytic products because they can be attached to anarray via these labels. Furthermore, secondary labels can be useful forseparating unreacted protease substrates or proteolytic products fromother components of a protease reaction, or detecting these moleculeswhen bound to probes, for example, in an array.

A label moiety can be attached to a protease substrate or other agentuseful in the invention using methods known in the art. For example, alabel moiety can be attached to a base, ribose, phosphate, or analogousstructure of a nucleic acid moiety. In particular embodiments, a moietycan be incorporated using modified nucleosides that are added to agrowing nucleotide strand, for example, during a detection step thatincludes modification of a probe or hybridized nucleic acid moiety, asset forth in further detail below. Nucleosides can be modified, forexample, at the base, phosphate or the ribose. Analogous structures canbe modified in a nucleic acid analog in order to attach a label moiety.

A method of the invention can further include a step of detectinghybridization of a probe to a target molecule such as a proteasesubstrate or proteolytic product. Depending upon the particularapplication of the invention, a probe-target hybrid can be detectedusing a direct detection technique, or alternatively anamplification-based technique. Direct detection techniques include thosein which the level of nucleic acids in probe-fragment hybrids providesthe detected signal. For example, in the case of a hybrid formed at aparticular array location, the signal from the location arising from theresident hybrid or one of its component nucleic acids can be detectedwithout amplifying the hybrid or its component nucleic acids.Alternatively, detection can include amplification of the probe ortarget or both to increase the level of nucleic acid that is detected.

As set forth below in the context of various exemplary detectiontechniques, a probe, nucleic acid moiety of a target molecule or bothcan be labeled. Furthermore, nucleic acids in a probe-target hybrid canbe labeled prior to, during or after hybrid formation and determinationof protease activity based on detection of such labels.

Generally, detection, whether direct or based on an amplificationtechnique, can be achieved by methods that perceive properties that areintrinsic to a nucleic acid or a label associated with the nucleic acid.Useful properties include, those that can be used to distinguishdifferent nucleic acids alone, or in combination with other methods,such as attachment of the nucleic acids to solid-phase supports.Exemplary properties upon which detection can be based include, but arenot limited to, mass, electrical conductivity, energy absorbance,fluorescence, magnetism, luminescence or the like.

Detection of fluorescence can be carried out by irradiating a proteasesubstrate, proteolytic product or label moiety with an excitatorywavelength of radiation and detecting emitted radiation by methods knownin the art and described for example in Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999). Afluorophore can be detected based on any of a variety of fluorescencephenomena including, for example, emission, excitation, fluorescenceresonance energy transfer (FRET) intensity, quenching, anisotropy orlifetime at one or more wavelengths. FRET can be used to identifyhybridization between a probe bearing a donor fluorophore and a targetbearing an acceptor fluorophore due to transfer of energy from theexcited donor to the acceptor. Donor and acceptor can be on target andprobe, respectively, as well. In either case, hybridization can bedetected as a shift in wavelength maximum, reduction of donor emissionor appearance of acceptor emission for the hybrid.

Other detection techniques that can be used to detect a proteasesubstrate or proteolytic product include, for example, mass spectrometrywhich can be used to perceive a molecule or complex based on its mass;surface plasmon resonance which can be used to perceive a molecule orcomplex based on binding or dissociation from a surface; absorbancespectroscopy which can be used to perceive a molecule or complex basedon the wavelength of the energy it absorbs; calorimetry which can beused to perceive a molecule or complex based on changes in temperatureof its environment upon binding or dissociation; electrical conductanceor impedance which can be used to perceive a molecule or complex basedon changes in its electrical properties or in the electrical propertiesof its environment; magnetic resonance which can be used to perceive amolecule or complex based on presence of magnetic nuclei; or other knownanalytic spectroscopic or chromatographic techniques.

In particular embodiments, a probe-target hybrid can be detected basedon the presence of the probe, target or both in the hybrid, withoutsubsequent modification of the hybrid species. For example, apre-labeled target can be identified based on presence of the label at aparticular array location where a complementary probe resides.

Alternatively or additionally, a nucleic acid probe can be modifiedwhile hybridized to a target and the modification detected in a methodof determining protease activity. Similarly, a nucleic acid moiety in atarget that is hybridized to a probe can be modified. In embodimentswherein one or more nucleotides is added by an enzyme having polymeraseor ligase activity, the probe or target nucleic acid moiety will have a3′ hydroxyl that is accessible. For example, a probe can be attached toa solid-phase support via its 5′ end or via another location on thenucleic acid such that the 3′ end is free for enzymatic modification. Ifdesired a protease substrate used in the invention can include a nucleicacid moiety that is attached to a protein via its 5′ end or via anotherportion of the nucleic acid such that the 3, end is free for enzymaticmodification. Exemplary methods that can be used to modify a nucleicacid, such as a probe or nucleic acid moiety, for detection inaccordance with the invention include, for example, those utilizing anextension assay, such as ASPE or SBE; a ligation assay such asoligonucleotide ligation; an assay including extension and ligation(such as the GoldenGate™ assay of Illumina, Inc.); invader assay; probecleavage; or pyrosequencing as described, for example, in U.S. Pat. No.6,355,431 B1 or US Pat. App. Pub. No. 04/0259100.

A plurality of probes can be used in a method of the invention therebyallowing for detection of a plurality of protease substrates orproteolytic products. Accordingly, the invention further provides amethod of determining activity of at least one protease. The method caninclude the steps of (a) providing a plurality of different substrateseach having a protein moiety attached to a nucleic acid moiety, whereinthe sequence of each nucleic acid moiety is unique to the sequence of adifferent protein moiety; (b) contacting the substrates with at leastone protease under conditions wherein the at least one proteasecatalyzes cleavage of the protein moieties, thereby producingproteolytic products wherein the nucleic acid moieties are separatedfrom the protein moieties; (c) contacting the proteolytic products withnucleic acid probes under conditions wherein the nucleic acid moietieshybridize to complementary sequences of the probes; and (d) detectinghybridization of the proteolytic products to the probes, therebydetermining activity of the at least one protease.

An advantage of the invention is that a plurality of protease reactionscan be carried out in a multiplex format. Thus, a method of theinvention can be used to determine activity for a plurality of proteasesfor one or more different substrates, to determine activity of one ormore protease for a plurality of different substrates, or to determinethe effects of one or more different inhibitors on one or moreproteases. Such a plurality of protease reactions can be carried outsimultaneously and in the same reaction vessel. However, if desired aplurality of protease assays can be carried out sequentially in the sameor different reaction vessels using a method of the invention.

A multiplex reaction can include at least about 2, 3, 4, 5, 8, 10, 15,20, 24, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 200, 300, 386, 400,500, 1000 or more different protease substrates or suspected proteasesubstrates. The number of proteases can also be multiplexed such that areaction includes at least about 2, 3, 4, 5, 8, 10, 15, 20, 24, 30, 40,48, 50, 60, 70, 80, 90, 96, 100, 200, 300, 386, 400, 500, 1000 or moredifferent proteases or suspected protease substrates. A multiplexreaction can include any combination of these exemplary numbers ofproteases and protease substrates. Furthermore, a multiplex reaction canbe carried out with fewer of each component if desired including, forexample, at most about a billion, million, 100,000, 10,000, 1,000, 500,386, 96, 48, 8 or 5 different proteases, proteases substrates or both(whether known or suspected).

Different protease substrates used in a method of the invention can havedifferent amino acid sequences such that identification of thosesequences that are cleaved by a protease, those that are notsubstantially cleaved by the protease, or both can be used to determinethe sequence specificity of the protease. In such embodiments, eachdifferent protein moiety can be attached to a nucleic acid moiety havinga particular nucleotide sequence. In this way different nucleotidesequences can be correlated with different amino acid sequences allowingconvenient identification of which protease substrates are cleaved ornot cleaved based on the identity of the nucleotide sequence. Forexample, the amino acid sequence of a protein-nucleic acid substratethat is recognized by a particular protease can be determined accordingto the sequence of a nucleic acid probe that hybridizes to the nucleicacid moiety of the substrate or its proteolytic product. An advantage ofcorrelating the nucleotide sequence with a particular protein moiety isthat the same label can be attached to several different substrates, forexample, in a multiplex protease assay, allowing a plurality of thesubstrates or their proteolytic products to be detected en masse usinguniform detector settings yet distinguished one from the other based onthe identity of the nucleic acid probe to which they hybridize. As setforth below, the components of a multiplexed protease assay can becontacted with an array of solid phase probes, and the componentsdistinguished based on detection of the same label at differentlocations in the array, thereby allowing determination of an activity ofone or more proteases in the reaction.

A plurality of nucleic acid probes used in a method of the invention canbe included in an array of probes attached to one or more solid support.In particular embodiments, probes useful in detecting proteasesubstrates, proteolytic products or other target molecules can beattached to particles that are arrayed or otherwise spatiallydistinguished. Exemplary particles include microspheres or beads. Itwill be understood that particles such as microspheres or beads can bespherical or approximately spherical but need not be perfectlyspherical. Rather solid phase particles having other shapes including,but not limited to, cylinders, disks, plates, chips, slivers orirregular shapes can be used. In addition, particles used in theinvention can be porous, thus increasing the surface area available forattachment or detection of molecules such as probes or targets. Particlesizes can range, for example, from nanometers such as about 100 nmbeads, to millimeters, such as about 1 mm beads, with particles ofintermediate size such as at most about 0.2 micron, 0.5 micron, 5 micronor 200 microns being useful. The composition of the beads can varydepending, for example, on the application of the invention or themethod of synthesis. Typically, useful particles consist of asubstantially non-compressible or inelastic material compared to abiological cell such as plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria solcarbon graphite, titanium dioxide, latex, Teflon™, cross-linked dextranssuch as Sepharose™, cellulose, or nylon. However, if desired abiological cell or similarly compressible particle such as across-linked micelle can be used as a solid phase support in theinvention. Other suitable bead compositions include, but are not limitedto, those used in peptide, nucleic acid and organic moiety synthesis orothers described, for example, in Microsphere Detection Guide from BangsLaboratories, Fishers Ind.

Several embodiments including the use of array-based detection in theinvention are exemplified below for beads or microspheres. Exemplarybead-based arrays that can be used in the invention include, withoutlimitation, those in which beads are associated with a solid supportsuch as those described in U.S. Pat. No. 6,355,431 B1, US 2002/0102578and PCT Publication No. WO 00/63437. Beads can be located at discretelocations, such as wells, on a solid-phase support, whereby eachlocation accommodates a single bead. Alternatively, discrete locationswhere beads reside can each include a plurality of beads as described,for example, in US Pat. App. Nos. US 2004/0263923, US 2004/0233485, US2004/0132205, or US 2004/0125424. Beads can be associated with discretelocations via covalent bonds or other non-covalent interactions such asgravity, magnetism, ionic forces, van der Waals forces, hydrophobicityor hydrophilicity. However, the sites of an array of the invention neednot be discrete sites. For example, it is possible to use a uniformsurface of adhesive or chemical functionalities that allows theattachment of particles at any position. Thus, the surface of an arraysubstrate can be modified to allow attachment or association ofmicrospheres at individual sites, whether or not those sites arecontiguous or non-contiguous with other sites. Thus, the surface of asubstrate can be modified to form discrete sites such that only a singlebead is associated with the site or, alternatively, the surface can bemodified such that a plurality of beads populates each site.

Beads or other particles can be loaded onto array supports using methodsknown in the art such as those described, for example, in U.S. Pat. No.6,355,431. In some embodiments, for example when chemical attachment isdone, particles can be attached to a support in a non-random or orderedprocess. For example, using photoactivatible attachment linkers orphotoactivatible adhesives or masks, selected sites on an array supportcan be sequentially activated for attachment, such that definedpopulations of particles are laid down at defined positions when exposedto the activated array substrate. Alternatively, particles can berandomly deposited on a substrate. In embodiments where the placement ofprobes is random, a coding or decoding system can be used to localizeand/or identify the probes at each location in the array. This can bedone in any of a variety of ways, for example, as described in U.S. Pat.No. 6,355,431 or WO 03/002979. A further encoding system that is usefulin the invention is the use of diffraction gratings as described, forexample, in US Pat. App. Nos. US 2004/0263923, US 2004/0233485, US2004/0132205, or US 2004/0125424.

An array of beads useful in the invention can also be in a fluid formatsuch as a fluid stream of a flow cytometer or similar device. Exemplaryformats that can be used in the invention to distinguish beads in afluid sample using microfluidic devices are described, for example, inU.S. Pat. No. 6,524,793. Commercially available fluid formats fordistinguishing beads include, for example, those used in xMAP™technologies from Luminex or MPSS™ methods from Lynx Therapeutics.

Any of a variety of arrays known in the art can be used in the presentinvention. For example, arrays that are useful in the invention can benon-bead-based. A particularly useful array is an Affymetrix® GeneChip®array. GeneChip® arrays can be synthesized in accordance with techniquessometimes referred to as VLSIPS™ (Very Large Scale Immobilized PolymerSynthesis) technologies. Some aspects of VLSIPS™ and other microarrayand polymer (including protein) array manufacturing methods andtechniques have been described in U.S. patent Ser. No. 09/536,841,International Publication No. WO 00/58516; U.S. Pat. Nos. 5,143,854,5,242,974, 5,252,743, 5,324,633, 5,445,934, 5,744,305, 5,384,261,5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681,5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711,5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659,5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601,6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846, 6,022,963,6,083,697, 6,291,183, 6,309,831 and 6,428,752; and in PCT ApplicationsNos. PCT/US99/00730 (International Publication No. WO 99/36760) andPCT/US01/04285.

A spotted array can also be used in a method of the invention. Anexemplary spotted array is a CodeLink™ Array available from AmershamBiosciences. CodeLink™ Activated Slides are coated with a long-chain,hydrophilic polymer containing amine-reactive groups. This polymer iscovalently crosslinked to itself and to the surface of the slide. Probeattachment can be accomplished through covalent interaction between theamine-modified 5′ end of the oligonucleotide probe and the aminereactive groups present in the polymer. Probes can be attached atdiscrete locations using spotting pens. Useful pens are stainless steelcapillary pens that are individually spring-loaded. Pen load volumes canbe less than about 200 nL with a delivery volume of about 0.1 nL orless. Such pens can be used to create features having a spot diameterof, for example, about 140-160 μm. In a preferred embodiment, nucleicacid probes at each spotted feature can be 30 nucleotides long. However,probes having other lengths such as those set forth elsewhere herein canalso be attached at each spot.

An array that is useful in the invention can also be manufactured usinginkjet printing methods such as SurePrint™ Technology available fromAgilent Technologies. Such methods can be used to synthesizeoligonucleotide probes in situ or to attach pre-synthesized probeshaving moieties that are reactive with a support surface. A printedmicroarray can contain 22,575 features on a surface having standardslide dimensions (about 1 inch by 3 inches). Typically, the printedprobes are 25 or 60 nucleotides in length. However, probes having otherlengths such as those set forth elsewhere herein can also be printed ateach location.

An exemplary high density array is an array of arrays or a compositearray having a plurality of individual arrays that is configured toallow processing of multiple samples. Such arrays allow multiplexdetection of protease assays. Exemplary composite arrays that can beused in the invention, for example, in multiplex detection formats aredescribed in U.S. Pat. No. 6,429,027 and U.S. Pat. App. Pub. No.2002/0102578. In particular embodiments, each individual array can bepresent within each well of a microtiter plate. Thus, depending on thesize of the microtiter plate and the size of the individual array, veryhigh numbers of assays can be run simultaneously; for example, usingindividual arrays of 2,000 probes and a 96 well microtiter plate,192,000 assays can be performed in parallel; the same number of probesin each well of a 384 microtiter plate yields 768,000 simultaneousassays, and in a 1536 microtiter plate gives 3,072,000 assays.

In some embodiments, solid-phase attached probes, such as nucleic acidsor peptides, can be synthesized by sequential addition of monomer unitsdirectly on a solid support used in an array such as a bead or slidesurface. Methods known in the art for synthesis of a variety ofdifferent chemical compounds on solid supports can be used in theinvention, such as methods for solid-phase synthesis of peptides,organic moieties, and nucleic acids. Alternatively probes can besynthesized first, and then covalently attached to a solid support, forexample, via reactive functional groups. Functionalized solid supportscan be produced by methods known in the art or, if desired, obtainedfrom any of several commercial suppliers. Exemplary surface chemistriesthat are useful in the invention include, but are not limited to, aminogroups such as aliphatic and aromatic amines, carboxylic acids,aldehydes, amides, chloromethyl groups, hydrazide, hydroxyl groups,sulfonates or sulfates. If desired, a probe can be attached to a solidsupport via a chemical linker. Such a linker can have characteristicsthat provide, for example, stable attachment, reversible attachment,sufficient flexibility to allow desired interaction with a targetmolecule to be detected, or to avoid undesirable binding reactions.Further exemplary methods that can be used in the invention to attachpolymer probes to a solid support are described in U.S. patentapplication Ser. Nos. 10/651,568 or 10/739,959; WO 01/41918; WO04/001646; Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026(1994); Khrapko et al., Mol Biol (Mosk) (USSR) 25:718-730 (1991);Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383 (1995) or Guoet al., Nucleic Acids Res. 22:5456-5465 (1994).

Very high density arrays are useful in the invention including, forexample, those having from about 10,000,000 array locations/cm² to about2,000,000,000 array locations/cm² or from about 100,000,000 arraylocations/cm² to about 1,000,000,000 array locations/cm². High densityarrays can also be used including, for example, those in the range fromabout 100,000 array locations/cm² to about 10,000,000 arraylocations/cm² or about 1,000,000 array locations/cm² to about 5,000,000array locations/cm². Moderate density arrays useful in the invention canrange from about 10,000 array locations/cm² to about 100,000 arraylocations/cm², or from about 20,000 array locations/cm² to about 50,000array locations/cm². Low density arrays are generally less than 10,000particles/cm² with from about 1,000 array locations/cm² to about 5,000array locations/cm² being useful in particular embodiments. Very lowdensity arrays having less than 1,000 array locations/cm², from about 10array locations/cm² to about 1000 array locations/cm², or from about 100array locations/cm² to about 500 array locations/cm² are also useful insome applications.

A solid-phase support used in an array of the invention can be made fromany material that can be modified to contain discrete individual sitesor to attach a desired probe. In embodiments where arrays of particlesare used, a material that is capable of attaching or associating withone or more type of particles can be used. Useful supports include, butare not limited to, glass; modified glass; functionalized glass;plastics such as acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon, or the like; polysaccharides; nylon;nitrocellulose; resins; silica; silica-based materials such as siliconor modified silicon; carbon; metal; inorganic glass; optical fiberbundles, or any of a variety of other polymers. Useful supports includethose that allow optical detection, for example, by being translucent toenergy of a desired detection wavelength and/or do not themselvesappreciably fluoresce at particular detection wavelengths.

The surface of a solid-phase support can include a plurality ofindividual arrays that are physically separated from each other. Forexample, physical separation can be due to the presence of assay wells,such as in a microtiter plate. Other barriers that can be used tophysically separate array locations include, for example, gaskets,raised barriers, channels, hydrophobic regions that will deter flow ofaqueous solvents or hydrophilic regions that will deter flow of apolaror hydrophobic solvents.

Arrays that are physically separated from each other provide separateassay locations. An assay location can include an array of probes andprovide a vessel for holding a fluid such that the fluid contacts theprobes. For example, an assay location containing a multiplex proteasereaction can be contacted with an array of probes under hybridizationconditions set forth herein or known in the art. Similarly, a wash fluidor fluid containing other reagents or analytes described herein can becontacted with an array of probes when placed in an assay location. Anassay location can be enclosed, if desired, for example, to form ahybridization chamber. Exemplary enclosures include, without limitation,a cassette, enclosed well, or a slide surface enclosed by a gasket ormembrane or both. Further exemplary enclosures that are useful in theinvention are described in WO 02/00336, US Pat. App. Pub. 02/0102578 orthe references cited previously herein in regard to different types ofarrays.

In a particular embodiment, an array support can be an optical fiberbundle or array, as is generally described in U.S. Ser. No. 08/944,850,U.S. Pat. No. 6,200,737; WO 98/40726; and WO 98/50782. Also useful inthe invention is a preformed unitary fiber optic array having discreteindividual fiber optic strands that are co-axially disposed and joinedalong their lengths. A distinguishing feature of a preformed unitaryfiber optic array compared to other fiber optic formats is that thefibers are not individually physically manipulable; that is, one strandgenerally cannot be physically separated at any point along its lengthfrom another fiber strand.

In a particular embodiment, several levels of redundancy can be builtinto an array used in the invention. As will be appreciated by those inthe art, there are at least two types of redundancy that can be builtinto an array: the use of multiple identical probes or the use ofmultiple probes directed to the same target, but having differentchemical functionalities. For example, for the detection of nucleicacids, sensor redundancy utilizes a plurality of sensor elements, suchas beads, having identical binding ligands. Target redundancy utilizessensor elements with different probes to the same target. For example,one probe can span the first 25 bases of a target, a second probe canspan the second 25 bases of the target, etc. As will be appreciated bythose in the art, the number of redundant probes in a sub-populationwill vary with the application and use of a particular array. Ingeneral, anywhere from 2 to thousands of redundant probes can be used,including, for example, about 5, 10, 20, 50 or 100 probes that are atdifferent locations but otherwise identical or capable of binding thesame target molecule.

Building redundancy into an array can give several non-limitingadvantages, including the ability to make quantitative estimates ofconfidence about the data collected from an array. Also redundancy canprovide substantial increases in sensitivity due to the ability to sumsignals. A variety of statistical mathematical analyses can be done foranalysis of large data sets. Exemplary analyses include, but are notlimited to, baseline adjustment, averaging, standard deviation analysis,distribution and cluster analysis, confidence interval analysis, meantesting, or the like. Analyses based on redundancy that can be used inthe invention are generally described in texts such as Freund andWalpole, Mathematical Statistics, Prentice Hall Inc., New Jersey (1980).Other methods for making and using redundant arrays are described, forexample, in U.S. Pat. No. 6,355,431 and WO 00/60332. Redundancy can beparticularly useful for increasing confidence levels or determiningstatistical validity for measurements of kinetic or thermodynamicproperties of protease activity such as binding constants, maximumvelocity, catalytic rate constant or others as described for example, inSegel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium andSteady-State Enzyme Systems, Wiley, John & Sons, Incorporated (1994).

Although the invention is exemplified above and elsewhere herein withrespect to an array of immobilized probes, those skilled in the art willrecognize that other detection formats can be employed as well. Forexample, the methods set forth herein can be carried out in solutionphase rather than solid phase. Accordingly, solution phase probes canreplace immobilized probes in the methods set forth herein. Solutionphase probes can be detected according to properties such as those setherein in regard to detection labels or detection moieties. For example,probes can have identifiable charge, mass, charge to mass ratio or otherdistinguishing properties. Such distinguishing properties can bedetected, for example, in a chromatography system including, forexample, capillary electrophoresis, acrylamide gel, agarose gel or thelike, or in a spectroscopic system such as mass spectroscopy.

A method of the invention can include a step of comparing the level of aprotease substrate with the level of a proteolytic product derived fromthe substrate in the presence of a particular protease. Such acomparison can be particularly useful in embodiments where proteaseactivity is indicated by a loss of signal. For example, if an assay isperformed in which protease activity is determined based on removal of alabel from a protease substrate then loss of signal can occur either dueto protease activity or due to an experimental artifact such as poorsignal detection, loss of substrate or the like. In this example,detection of the level of protease substrate can be used as a controlfor experimental artifact by comparing to the level of proteolyticproduct. Such comparisons can be carried out for pluralities ofproteases, protease substrates or proteolytic products or a combinationthereof. Thus, a plurality of comparisons can be made with a pluralityof protease substrates in accordance with methods set forth herein.

As set forth previously herein, a plurality of different proteasesubstrates useful in a method of the invention can have substantiallythe same label. In such cases, target molecules can be distinguishedbased on detection of the label in combination with identification ofthe nucleotide sequence of the nucleic acid moieties. Such methods areexemplified above based on the use of arrays in which a plurality oftarget molecules are detected based on signals from the labels and thenucleotide sequence of each is identified based on the location of thetarget molecules in the array. However, it is also possible to use oneor more different labels for a plurality of protease substrates orproteolytic products used in a method of the invention. Accordingly, aparticular target molecule can be identified within a plurality ofdifferent target molecules based on the identity of the label alone.Thus, an array need not be used to differentiate a plurality of targetmolecules in a method of the invention, for example, if a sufficientnumber of different labels are available to uniquely identify eachtarget molecule.

A multiplex reaction can be carried out under conditions wherein aplurality of different proteases are present and one or more of theproteases is capable of inactivating or inhibiting one or more otherproteases present in the same reaction vessel. In such embodiments,control reactions can be carried out in which one or more of theproteases are omitted. Comparison of the protease activity of ananalytical reaction containing a full set of proteases with a controlreaction omitting one or more of the proteases can be used to evaluatecross reactivity between proteases. Furthermore, one or more proteaseinhibitors can be added in sufficient concentration to inactivate one ormore proteases that are present in a reaction or potentially present ina reaction. Such protease inhibitors can be added to prevent unwantedproteolysis of a protease under investigation or its substrate.Alternatively, varying concentrations of a protease inhibitor can beused to determine if protease cross reactivity is occurring.

The invention further provides for methods in which protease activitycan be determined as a gain of signal. For example, the inventionprovides a method of determining activity of a protease including thesteps of (a) providing a substrate having a protein moiety attached to anucleic acid moiety and a ligand moiety; (b) contacting the substratewith a protease under conditions wherein the protease catalyzes cleavageof the protein moiety, thereby producing a proteolytic product whereinthe nucleic acid moiety is separated from the protein moiety and theligand moiety; (c) contacting the proteolytic product with a receptorunder conditions wherein the ligand moiety binds to the receptor to forma complex; (d) separating the complex from the nucleic acid moiety,thereby forming a separation product having the nucleic acid moiety; (e)contacting the separation product with a probe nucleic acid underconditions wherein the nucleic acid moiety hybridizes to a complementarysequence of the probe; and (f) detecting hybridization of the separationproduct to the probe, thereby determining activity of the protease.

An exemplary embodiment wherein a proteolytic product is detectedfollowing separation from protease substrate is shown in FIG. 6. Theprotease substrate shown in the figure has a biotin attached to theamino terminal end of the protein moiety and the carboxy terminal end ofthe protein moiety is attached to the 5′ end of a DNA moiety.Additionally, the 3′ end of the DNA moiety is attached to a FAM label.Protease activity results in cleavage of the protein moiety such thatthe biotin moiety is separated from the FAM labeled DNA moiety. If theprotease substrate is not cleaved then the DNA moiety remains attachedto the biotin moiety. Support bound streptavidin when contacted with thereaction mixture can bind to the biotinylated protease substrate and theportion of the proteolytic product that lacks the FAM labeled DNAmoiety. However, the proteolytic product that has the FAM labeled DNAmoiety does not bind to the streptavidin, thereby allowing it to beseparated from the uncleaved protease substrate and the other product.The separated proteolytic product is contacted with a solid-phase probehaving a sequence that is complementary to the DNA moiety and proteaseactivity is determined according to the presence of FAM signal on theprobe. It will be understood that the method exemplified in FIG. 6 canbe used with a protease substrate having the nucleic acid moiety, labelor both attached at different locations from those shown. In suchembodiments, the substrate and product can be distinguished so long asprotease activity separates the nucleic acid moiety from the labelmoiety used for solid phase removal of uncleaved substrate. Furthermore,another nucleic acid moiety, label (in place of FAM or biotin) orcombination thereof can be used in a protease substrate having theconfiguration exemplified in FIG. 6. Examples of other moieties that areuseful are set forth previously herein.

Any of a variety of secondary labels can be used to separate proteasesubstrates from proteolytic products. Examples are set forth hereinpreviously. The secondary labels can be attached to a solid-phasesupport such as a particle or array or other supports set forth hereinpreviously. In further embodiments, a solution-phase primary label orsolution-phase secondary label can be used for separating a proteolyticproduct from a protease substrate. For example, a solution phase labelcan be used to monitor separation of the substrate and product in aseparation method such as chromatography, flow sorting, electrophoresis,extraction or others known in the art as described, for example, inScopes, Protein Purification 3^(rd) Ed. Springer Verlag New York (1994).

The invention further provides a method in which protease activity canbe determined as a gain of signal for a plurality of proteasesubstrates. The method can include the steps of (a) providing aplurality of protease substrates each having a protein moiety attachedto a nucleic acid moiety and a ligand moiety, wherein the sequence ofeach nucleic acid moiety is unique to the sequence of a differentprotein moiety; (b) contacting the protease substrates with at least oneprotease under conditions wherein the at least one protease catalyzescleavage of the protein moieties, thereby producing proteolytic productswherein the nucleic acid moieties are separated from the proteinmoieties and the ligand moieties; (c) contacting the proteolyticproducts with at least one receptor under conditions wherein the ligandmoieties bind to the at least one receptor to form complexes; (d)separating the complexes from the nucleic acid moieties, thereby formingseparation products comprising the nucleic acid moieties; (e) contactingthe separation products with nucleic acid probes under conditionswherein the nucleic acid moieties hybridize to complementary sequencesof the probes; and (f) detecting hybridization of the separationproducts to the probes, thereby determining activity of the at least oneprotease.

In embodiments wherein a plurality of proteolytic products are detectedfollowing separation from protease substrates, separation can bemediated by substantially the same ligand moiety attached to eachprotease substrate. Thus, a receptor, or a group of receptors havingaffinity for substantially the same ligand, can be used for separationof the plurality of protease products from the protease substrates fromwhich they were derived. For example, a receptor can be used to carryout separation in a format wherein a plurality of proteolytic productsis present simultaneously in the same tube. The use of the same receptorfor a plurality of proteolytic products can provide for more efficientseparation in such multiplex formats. However, the use of substantiallythe same ligand for a plurality of proteolytic products can also be usedin formats where proteolytic products are individually separated fromthe protease substrates. Exemplary formats include, but are not limitedto, individual assays or multiple assays performed sequentially.

A plurality of different proteolytic products can be separated fromother reaction components based on the use of different ligands. Thedifferent ligands can have affinity for the same receptor or fordifferent receptors. For example, biotin and any number of differentbiotin analogs having affinity for avidin or streptavidin can beattached to different proteolytic products and will behave assubstantially the same ligand when used avidin-based orstreptavidin-based separation. Alternatively the use of differentligands having affinity for different receptors can be used tosubfractionate different protease substrates or proteolytic products.For example, different subpopulations of protease substrates orproteolytic products can each be attached to different ligands and thedifferent subpopulations can be separated from each other usingdifferent receptors having specificity for the different ligands.

The methods set forth herein can be used to identify one or moreinhibitors for a protease. A known or putative inhibitor can be includedin a protease reaction such as those exemplified herein. The activity ofthe inhibitor can be determined using analysis methods known in the artincluding, for example, comparison to a similar protease reactioncarried out in the absence of the inhibitor or enzyme kinetic methodssuch as those set forth above. The invention is particularly useful fordetermining the activity for a plurality of protease inhibitors. Forexample, the activity for a plurality of protease inhibitors can bedetermined in a multiplex assay carried out in a single tube andincluding at least one protease and at least one protease substrate. Amultiplex reaction can include at least about 2, 3, 4, 5, 8, 10, 15, 20,24, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 200, 300, 386, 400, 500,1000 or more different protease inhibitors. Furthermore, a multiplexreaction can be carried out with fewer protease inhibitors if desiredincluding, for example, at most about a billion, million, 100,000,10,000, 1,000; 500; 386, 96, 48, 8 or 5 different protease inhibitors.Thus, the invention can be used to screen for new protease inhibitorsand to identify an inhibitor having specificity for a particularprotease, group of proteases or family of proteases.

A protease assay of the invention can be used to characterize abiological system such as a cell, tissue, organism or group oforganisms. For example, the protease activity for one or more proteasesin a first system can be determined and, if desired, can be compared tothe protease activity of a second biological system or to a referenceprotease activity. It will be understood that, in this regard, theprotease activity for a biological system can be due to one or moreproteases. Accordingly, the invention can be used to identify theprotease complement present in a particular biological system. Forexample, a biological system can produce a particular signature ofproteolytic activities toward a plurality of protease substrates thatindicates the identity of the protease complement. As a further example,a method of the invention can be used to separately evaluate theprotease activity of a biological system toward one or more individualprotease substrates.

Exemplary biological systems that can be used in a method of theinvention include, without limitation, a mammal such as a rodent, mouse,rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat,dog, primate, human or non-human primate; a plant such as Arabidopsisthaliana, corn (Zea mays), sorghum, oat (oryza sativa), wheat, rice,canola, or soybean; an algae such as Chlamydomonas reinhardtii; anematode such as Caenorhabditis elegans; an insect such as Drosophilamelanogaster, mosquito, fruit fly, honey bee or spider; a fish such aszebrafish (Danio rerio); a reptile; an amphibian such as a frog orXenopus laevis; a dictyostelium discoideum; a fungi such as pneumocystiscarinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae orSchizosaccharomyces pombe; or a plasmodium falciparum. A method of theinvention can also be used to detect protease activity for a prokaryotesuch as a bacterium, Escherichia coli, staphylococci or mycoplasmapneumoniae; an archae; a virus such as Hepatitis C virus or humanimmunodeficiency virus; or a viroid. A homogeneous culture or populationof the above organisms can be evaluated using the invention as can acollection of several different organisms, for example, in a communityor ecosystem.

A cell from which one or more proteases is obtained for use in theinvention can be a normal cell or a cell displaying one or more symptomof a particular disease or condition. Thus, a protease sample used in amethod of the invention can be obtained from a cancer cell, neoplasticcell, necrotic cell, cell experiencing an auto-immune condition,apoptotic cell or the like. Those skilled in the art will know or beable to readily determine methods for isolating one or more proteasefrom a cell, bodily fluid or tissue using methods known in the art suchas those described in Scopes, supra (1994) or Coligan et al., supra(2000). In particular embodiments of the invention, a crude cell lysatecontaining a collection of protease can be directly detected in a methodof the invention without further isolation of the proteases.Alternatively, one or more proteases can be further isolated from othercellular components prior to protease assay. Proteases can be isolatedusing known methods including, for example, liquid phase extraction,precipitation, solid phase extraction, chromatography, centrifugation orthe like. Such methods are described, for example, in Barrett et al.,supra (2004), Scopes, supra (1994) or Coligan et al., supra (2000).

A method of the invention can further include steps of isolating aparticular type of cell or tissue. Exemplary methods that can be used ina method of the invention to isolate a particular cell from other cellsin a population include, but are not limited to, Fluorescent ActivatedCell Sorting (FACS) as described, for example, in Shapiro, PracticalFlow Cytometry, 3rd edition Wiley-Liss; (1995), density gradientcentrifugation, or manual separation using micromanipulation methodswith microscope assistance. Exemplary cell separation devices that areuseful in the invention include, without limitation, a Beckman JE-6™centrifugal elutriation system, Beckman Coulter EPICS ALTRA™computer-controlled Flow Cytometer-cell sorter, Modular Flow Cytometer™from Cytomation, Inc., Coulter Counter™ or Channelyzer™ system, densitygradient apparatus, Cytocentrifuge, Beckman J-6™ centrifuge, EPICS V™dual laser cell sorter, or EPICS PROFILE™ flow cytometer. A tissue orpopulation of cells can also be removed by surgical techniques. Forexample, a tumor or cells from a tumor can be removed from a tissue bysurgical methods, or conversely non-cancerous cells can be removed fromthe vicinity of a tumor.

The invention can be used for diagnosis or prognosis of a disease orcondition. For example, the protease activity for a test cell or tissuethat is known or suspected of being affected by a particular disease orcondition can be determined using a method of the invention. If desired,protease activity can also be determined for a second cell or tissuethat serves as a control and the results from the control cell or tissuecompared to the results from the test cell or tissue. A control cell ortissue can be derived from a non-affected cell or tissue from the sameindividual as the test cell or tissue. Alternatively, the control cellor tissue can be obtained from a separate individual. The separateindividual can be a non-affected individual that is related to the testindividual within one, two, three or more generations. Alternatively,the separate individual can be effectively unrelated being manygenerations removed, or even of a different ethnicity. In some cases itmay be useful to use a control individual having similar ethnicity asthe test individual.

Protease activity determined in a method of the invention for aparticular biological system can be correlated with one or more symptomsof a disease or condition. Those skilled in the art will know or be ableto determine symptoms that are indicative of a disease or conditionbeing evaluated. An exemplary reference describing symptoms forparticular diseases or conditions is The Merck Manual of Diagnosis andTherapy 16th Ed., Edited by Berkow, Published by Merck and Co., Inc.,Rahway N.J. (1992).

A method of the invention can also be used to evaluate the effect of aparticular treatment on a biological system. For example, a biologicalsystem can be treated with a particular drug or agent suspected ofhaving an effect on the system and protease activity measured for thetreated system. The drug or agent can act, for example, as a proteaseinhibitor or activator, can act to increase protease expression, or canact to destabilize one or more protease, thereby reducing half life inits natural milieu. Comparison can be made to the protease activity fora control system that has not been treated or that has been treated to adifferent extent. Thus, protease activity as measured by a method of theinvention can be used to evaluate dose response, efficacy, time periodof response or the like for a biological system undergoing a particulartreatment. If available, comparison can be made to a reference activity,for example, as stored in a database or other storage medium.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Evaluating Protease Activity Using a Protein-DNA Conjugate asSubstrate

This example demonstrates a singleplex assay in which protease activityis detected based on cleavage of a nucleic acid-protein conjugate.

Caspase-3 protease activity was carried out as follows. Caspase-3protease was incubated with a caspase-3 substrate having theconfiguration shown in FIG. 2. Briefly, the caspase-3 substrate includeda protein moiety (peptide) having a caspase-3 amino acid recognitionsequence. The protein moiety was attached at its amino terminus to aFAM-labeled DNA moiety and at its carboxy terminus to a biotin. Theenzymatic reactions were performed in 30 to 60 microliter volumes havingsubstrate concentrations in the range of 20 nM to 150 nM. The enzymeconcentrations ranged from a few ng/ml to a few mg/ml. The assay bufferwas 50 mM Tris-HCL, pH 8.0, 1 mM CaCl₂ and 0.1% Tween-20 (v/v). Theassay was carried out at 37° C. at varying times from 5 minutes totwelve hours.

Following incubation of the protease and substrate, the reaction mixturewas extracted with streptavidin beads provided in molar excess for 15minutes to remove uncleaved substrate (and cleaved protein fragmentslacking the DNA moiety). Following removal of the streptavidin beads,the remaining mixture, containing FAM-labeled DNA fragments of thecleaved caspase-3 substrate, was diluted into GoldenGate™ hybridizationbuffer (Illumina, Inc., San Diego, Calif.) and hybridized onto aSentrix® BeadArray Matrix for one hour at room temperature. TheBeadArray matrix included nucleic acid probes that were complementary tothe FAM-labeled DNA fragments. The array was imaged using the UltraImager™ (Ultra-Lum, Inc., Claremont, Calif.) set at 485 nm excitationand 535 nm emission to detect the presence of FAM on the probes.

A titration was carried out using protease reactions in which caspase-3substrate was incubated with various concentrations of caspase-3 enzyme(from 1 micromolar to 0.01 nanomolar). A first control (No E, No SA) wasevaluated using the protease assay conditions set forth above exceptthat caspase-3 enzyme was absent and streptavidin extraction wasomitted. The signal intensity resulting for the No E, No SA controlindicates the maximum amount expected from the FAM present in a proteaseassay. A second control reaction (No E) was evaluated using conditionsset forth above except that caspase-3 enzyme was absent. In this casethe streptavidin extraction step is included. Thus, the No E controlrepresents the minimum or background signal expected from a proteaseassay.

As shown in FIG. 7, mean intensity from probes complementary to the DNAmoiety was above 5000 counts for the No E, No SA control, whereas lessthan 500 counts were measured for the No E control. Titration ofcaspase-3 enzyme indicated that nearly complete proteolysis of thecaspase-3 substrate occurred in the presence of 1 micromolar, 100nanomolar, 10 nanomolar and 1 nanomolar caspase-3 enzyme. However, asthe concentration of caspase-3 enzyme was reduced to 0.1 nanomolar and0.01 nanomolar the extent of proteolysis was also reduced.

The BeadChip arrays include multiple probes for each DNA moiety. Anadvantage of using multiple probes is that statistical analysis can becarried out on the detected signals to increase confidence in theresults. Such statistical analysis can include standard deviationanalysis (as represented by the error bars in FIG. 7). Other statisticalanalyses known in the art can also be used, as desired.

Titration of caspase-3 substrate was carried out in the protease assay.As shown in FIG. 8, mean intensity for proteolyzed substrate wascomparable to intensity of the No E, No SA control at each concentrationtested, thereby indicating high sensitivity of the assay. C31E, C32E andC33E in the figure refers to serial dilutions of the caspase at half logintervals in the range of 300 to 17 nanomolar concentration.

FIG. 9 shows a plot of mean signal intensity measured for separateassays run for various times as indicated on the x-axis. Three separatecurves are presented for the three different concentrations of caspase-3substrate that were evaluated. The results of FIG. 9 indicate thatproteolysis was saturable for all three concentrations of caspase-3substrate when reacted with caspase-3 enzyme for increasing time periodsfrom 2 to 40 minutes.

An inhibition analysis was carried out in which the protease assay wasperformed in the presence of various concentrations of the Caspase-3inhibitor ZVAD-FMK (EMD Biosciences, Catalog #627610). FIG. 10 shows aplot of mean signal intensity vs. time for end point assays run in theabsence of inhibitor or in the presence of various concentrations ofZVAD-FMK. Comparison of the curve obtained in the absence of inhibitorwith those obtained in the presence of inhibitor indicates that as theconcentration of inhibitor was increased the extent of inhibition wasalso increased.

These results indicate that protease activity can be evaluated todetermine substrate specificity and inhibitor specificity using a methodin which a protein-DNA conjugate is cleaved, unreacted nucleicacid-protein conjugate is extracted based on a first label attached tothe protein moiety and, detection occurs based on a second labelattached to the DNA moiety

EXAMPLE II Multiplex Evaluation of Protease Activity on a Microarray

This example demonstrates a multiplex assay in which protease reactionsfor three different proteases with three different nucleic acid-proteinconjugates are carried out in simultaneous reactions and in a commonreaction vessel under conditions wherein each reaction can beindividually evaluated.

Protease assays were carried out as set forth above in Example I exceptthat each reaction included the three substrates 38-K, 40-MMP-2 and41-TEV. Each substrate had a configuration similar to that shown in FIG.2, including a protein moiety having a protease recognition sequence(for Kallikrein protease, MMP-2 protease, or Tobacco Etch Virus (TEV)protease, respectively) and a DNA moiety having a target sequence thatcomplements a unique probe of a Sentrix® BeadArray. Three separatesingle-plex assays were carried out each having one of the threeproteases.

The results of the single-plex assays are shown in FIG. 11. The bargraphs show mean signal intensity detected at each set of probes thatbind to the DNA moiety of a particular substrate (as listed on x-axis).Light grey bars represent signal obtained from complete reactions anddark grey bars represent signal obtained from control reactions lackingadded protease. As indicated by the plots, each enzyme was specific fora single one of the three substrates under the conditions used.

A three-plex reaction was carried out as set forth above for thesingle-plex reactions except that all three proteases were present inthe same reaction. The results obtained from a single array used todetect the reaction products are shown in FIG. 12. The results indicatedthat the three reactions could be individually evaluated based on acomplex reaction mixture detected on the same array.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the invention. Accordingly, the inventionis limited only by the claims.

1-24. (canceled)
 25. A protease substrate comprising a molecule having aprotein moiety, a nucleic acid moiety and a label moiety, wherein saidprotein moiety comprises a protease recognition sequence capable ofbeing cleaved by a protease, and wherein said label moiety is attachedin a configuration that allows said label moiety to be separated from aportion of said protein moiety upon proteolysis of said protein moiety.26. The composition of claim 25, wherein said label moiety comprises aligand.
 27. The composition of claim 25, wherein said label moiety isattached to said nucleic acid moiety.
 28. The composition of claim 25,wherein said label moiety is attached to said protein moiety
 29. Thecomposition of claim 25, wherein said protein moiety comprises a carboxyor amino terminal residue that is attached to said nucleic acid moiety.30. The composition of claim 25, wherein said protein moiety is attachedto the 3′ end or 5′ end of said nucleic acid moiety.
 31. The compositionof claim 25, wherein said protein moiety comprises an internal residuethat is attached to said nucleic acid moiety.
 32. The composition ofclaim 25, wherein said protein moiety is attached to an internalnucleotide of said nucleic acid moiety.
 33. The composition of claim 25,wherein attachment of said protein moiety to said nucleic acid moiety ismediated by a linker that comprises said label moiety.
 34. Thecomposition of claim 25, further comprising a second label moietyattached to said portion of said protein moiety that is separated fromsaid label moiety upon proteolysis.
 35. The composition of claim 34,wherein said label moiety is attached to said nucleic acid moiety andsaid second label moiety is attached to said protein moiety.
 36. Thecomposition of claim 34, wherein said label moiety is attached to afirst location of said protein moiety and said second label moiety isattached to a second location of said protein moiety.
 37. A compositioncomprising a plurality of different protease substrates, wherein eachdifferent protease substrates comprises a molecule having a differentprotein moiety covalently attached to a nucleic acid moiety and a labelmoiety, wherein the nucleotide sequence of said nucleic acid moiety isunique to the amino acid sequence of said different protein moiety,wherein said protein moiety comprises a protease recognition sequencecapable of being cleaved by a protease, and wherein said label moiety isattached in a configuration that allows said label moiety to beseparated from a portion of said protein moiety upon proteolysis of saidprotein moiety.
 38. The composition of claim 37, wherein said labelmoiety is the same for said different protease substrates.
 39. Thecomposition of claim 37, wherein said label moiety is different for saiddifferent protease substrates.
 40. The composition of claim 37, whereinsaid label moiety comprises a ligand.
 41. The composition of claim 37,wherein said label moiety is attached to said nucleic acid moiety. 42.The composition of claim 37, wherein said label moiety is attached tosaid protein moiety
 43. The composition of claim 37, wherein saidprotein moiety comprises a carboxy or amino terminal residue that isattached to said nucleic acid moiety.
 44. The composition of claim 37,wherein said protein moiety is attached to the 3′ end or 5′ end of saidnucleic acid moiety.
 45. The composition of claim 37, wherein saidprotein moiety comprises an internal residue that is attached to saidnucleic acid moiety.
 46. The composition of claim 37, wherein saidprotein moiety is attached to an internal nucleotide of said nucleicacid moiety.
 47. The composition of claim 37, wherein attachment of saidprotein moiety to said nucleic acid moiety is mediated by a linker thatcomprises said label moiety.
 48. The composition of claim 37, furthercomprising a second label moiety attached to said portion of saidprotein moiety that is separated from said label moiety uponproteolysis.
 49. The composition of claim 48, wherein said label moietyis attached to said nucleic acid moiety and said second label moiety isattached to said protein moiety.
 50. The composition of claim 48,wherein said label moiety is attached to a first location of saidprotein moiety and said second label moiety is attached to a secondlocation of said protein moiety.
 51. The composition of claim 48,wherein said second label moiety is the same for said different proteasesubstrates.
 52. The composition of claim 48, wherein said second labelmoiety is different for said different protease substrates.